1. Field of the invention
The invention relates to improved compositions and methods for the digestion of nucleic acids, specifically, to improved compositions and methods for cleaving base pair mismatches in double-stranded nucleic acid targets. More particularly, the invention concerns novel components of the reaction mixture in which RNase is used to cleave nucleic acids, and in particular base pair mismatches.
2. Description of the Art
The digestion of nucleic acids has important applications in the field of molecular biology. For example, digestion of single base mismatches in RNA/DNA and RNA/RNA targets has been used to detect mutations.
Nucleases are enzymes used to digest nucleic acid. DNases degrade single and/or double-stranded DNA, RNases degrade single and/or double-stranded RNA, and non-specific nucleases such as SI degrade both RNA and DNA, with preference for single-stranded nucleic acid. Nucleases are enzymes that catalyze the hydrolytic cleavage of a polynucleotide chain by cleaving the phosphodiester linkage between nucleotide residues. They can be classified as either exonucleases, which cleave nucleotides from the end of the chain, or endonucleases, which cleave from within the chain, and may specifically cleave single stranded or double stranded nucleic acids or both. Nucleases may also act only or preferentially on DNA, DNases, or RNA, RNases, or they may cleave both. Most nucleases cleave the nucleic acids without sequence specificity. However, some nucleases cleave specifically in a particular base or a specific sequence, such as restriction enzymes.
The use of nucleases to digest or cleave nucleic acids is well known in the field of molecular biology. For example, nucleases can be used to digest base pair mismatches that result from point mutations in genes. In the case of detecting mutations by digestion or cleavage the only nucleases which have been widely used are single-strand specific RNases; in particular, RNase A has been used for this purpose.
Methods for rapidly, reliably, and inexpensively detecting new point mutations have wide application in diagnosis and treatment of genetic diseases and cancer, and also in genetic counseling. These methods are of great benefit as well in basic research into the causes of a variety of human genetic diseases and in establishing human genetic linkage maps.
In most genetic diseases, the causative mutations are widely distributed over a large number of sites. Relatively few genetic disorders are caused by defined mutations at single sites; sickle cell anemia is one example of a disease phenotype that is always caused by the same specific mutation (i.e., an A-&gt;T transversion at codon 6 of the beta-globin gene). More commonly, genetic diseases, especially cancer, are associated with a number of different mutations in different sites in different arrays of genes. An example of this is breast cancer. For instance, mutations in the recently identified BRCA1 gene, which is thought to be associated with familial breast cancer, are scattered throughout a 5 kilobase coding region. Dispersed mutations in several other genes, including p53, EGFR, IGR, and her2/neu, are also believed to play important roles in breast cancer.
Methods for genetic screening by identifying mutations associated with most genetic diseases and cancer must be able to assess large regions of the genome. Once a relevant mutation has been identified in a given patient, other family members and affected individuals can be screened using methods which are targeted to that site. The ability to detect dispersed point mutations is critical for genetic counseling, diagnosis, and early clinical intervention as well as for research into the etiology of cancer and other genetic disorders. The ideal method for genetic screening would quickly, inexpensively, and accurately detect all types of widely dispersed mutations in genomic DNA, cDNA, and RNA samples, depending on the specific situation. Currently there are no methods which achieve these goals.
Historically, a number of different methods have been used to detect point mutations, including denaturing gradient gel electrophoresis ("DGGE"), restriction enzyme polymorphism analysis, chemical and enzymatic cleavage methods, and others (Cotton, 1989). The more common procedures currently in use include direct sequencing of target regions amplified by PCR and single-strand conformation polymorphism analysis ("SSCP").
Direct sequencing of PCR products is considered to be the most reliable method for identifying new mutations. However, sequencing is also the most expensive and labor-intensive genetic screening method. Direct sequencing is typically the most time-consuming step in the identification of point mutations, even with the advent of automated sequencing methods. Further, even DNA sequencing may not give a clear indication of a point mutation in some cases, for example when an individual is heterozygous for that allele. Non-specific background signals are often present in sequencing reactions, appearing as coincident bands on the sequencing ladder in manual sequencing methods using radioisotopes (Cheng and Haas, 1992), or appearing as non-specific fluorescent peaks in automated sequencing reactions. The main causes of this type of background are premature termination during the extension reaction and non-specific priming. Due to these limitations, and to the time and expense involved in sequencing large regions of DNA, direct sequencing is more practical as a tool to identify the specific nucleotide alterations in samples known to contain mutations, rather than as a primary screening method to assess large regions of the genome. Therefore, preliminary screening methods are needed to identify samples that contain mutations, and avoid the unnecessary labor, expense, and time needed for sequencing samples which do not contain mutations.
The most common screening method currently in use is SSCP. This method involves amplification of target regions, usually less than 300 bp long, which are denatured and separated on thin, native polyacrylamide gels. Point mutations are detected as mobility differences between wild-type controls and experimental samples. One drawback of SSCP is the requirement for radiolabeled material for analysis, due to the small mass amounts of the double-stranded DNA samples that must be used to prevent reannealing of the complementary strands after denaturation. Another disadvantage of SSCP is that the gels typically require long running periods (6-18 hours or longer) at high voltages, usually in a cold room with recirculation of the running buffer. These electrophoresis parameters are awkward, labor-intensive, hazardous, and require the use of expensive and specialized equipment. In addition, no single, optimal electrophoresis condition or gel composition for detection of mutations by SSCP, has yet been discovered. Therefore, each sample is typically assessed on multiple gels (for example, with and without 10% glycerol, or at room temperature and 4.degree. C.). Further, as the size of the target region assessed by SSCP increases, the detection rate decreases. For example, in one study, the detection rate decreased to 57% in 307 bp targets (Sarker et al., 1992). Therefore, SSCP is not effective for screening large regions of the genome in a single step.
Another method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA and RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single and multiple base point mutations. U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single-stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. After the RNase cleavage reaction, the RNase can be inactivated by proteolytic digestion and organic extraction, and the cleavage products are denatured by heating and analyzed by electrophoresis on denaturing polyacrylamide gels. For the analysis of cleavage products, the single-stranded products of the RNase A treatment are electrophoretically separated according to size and compared to similarly treated control duplexes. Currently available RNase mismatch cleavage assays, including those performed according to U.S. Pat. No. 4,946,773, require the use of radiolabeled RNA probes. The use of radiolabeled probes has many drawbacks, including the expense of the isotope, the time delay required for film exposure, and, particularly, the hazard radioisotopes present to workers during their synthesis, purification, and use at close range in the assay. The problems and costs associated with disposal of radioactive waste are also serious and well-documented. Further drawbacks of RNase mismatch cleavage assays in their present form, include the fact that only about one half to two thirds of point mutations are detected (Myers et al., 1985; Theophillus et al., 1989; Grompe, 1993). In light of these limitations, RNase mismatch cleavage assays have largely fallen into disuse.
The inventor has recently filed a U.S. patent application (Application No. 08/371,531, incorporated herein by reference) disclosing an RNase protection assay in which RNA transcripts of test sample and controls are produced by in vitro transcription of PCR products containing opposable T7 and SP6 phage promoter sequences. No radioisotopes are required in the disclosed method, termed non-isotopic RNase cleavage assay ("NIRCA.TM."), since the dual amplification steps of PCR and in vitro transcription yield large amounts (several micrograms) of target substrate, which permits the cleavage products to be visualized directly under UV light, when stained with ethidium bromide.
Sensitivity (i.e., the ability to detect 100% of all mutations) and specificity (lack of false positives) are important requirements for widespread acceptance of any new method for genetic screening. Even though the NIRCA.TM. method is a considerable improvement over past systems, there is still a drawback in that not all point mutations are detected by NIRCA.TM.. For example, while 90% of 30 point mutations were detected in an X-linked gene in the initial controlled study reported in the 08/371,531 application (along with two false positives in the 19 normal controls), the detection rate in a heterozygous system is likely to be less. In a clinical/diagnostic setting, where the majority of samples are normal in any give locus, the occurrence of even a low percentage of false positives could result in higher than desirable error rates. Of greater concern in clinical settings is the occurrence of false negatives, since overlooked mutations may result in improper diagnosis, treatment, and counseling. The reduced sensitivity of NIRCA.TM., relative to more laborious and expensive screening methods (e.g., SSCP, DGGE, direct sequencing) is its major disadvantage. A method to improve the sensitivity of the NIRCA.TM. assay would increase its utility for mutation detection and genetic analysis in many areas of basic and applied research, as well as in clinical/diagnostic settings.
Myers and Maniatis in U.S. Pat. No. 4,946,773 describe the detection of base pair mismatches using RNase A. Non-specific cleavage seen with RNase A has been a significant problem (Theophillus et al., 1989; Maniatis et al., U.S. Pat. No. 4,946,773). RNase A levels needed for optimum cleavage of mismatches are so high as to cause significant non-specific cleavage in the no-mismatch controls. Non-specific cleavage by RNase I has also been reported to be a problem when using this enzyme for mismatch detection. In the only currently known example of a method that employs RNase I for mismatch cleavage, the amount of RNase I used to cleave mismatches results in partial degradation of the base paired duplex. (Promega Technical Manual, 1994). Non-specific cleavage of the double-stranded duplex makes interpretation of the data more difficult and error-prone. Indeed, the two false-positive samples the inventor misidentified in an initial controlled study may be attributed to the high background of non-specific cleavage products.
Subsequent to the issue of the Myers and Maniatis patent, the E. coli enzyme, RNase I, which had been characterized as having a broader substrate specificity, was tested by the inventor for use in mismatch assays. This suggests that RNase I is a desirable enzyme to employ in the detection of base pair mismatches if components can be found to decrease the extent of non-specific cleavage and increase the frequency of cleavage of mismatches.
Unfortunately, the use of RNase I has proven difficult. The use of RNase I for mismatch detection is described in literature from Promega Biotech (Ekenberg and Hudson, 1994). Promega markets a kit containing RNase I that is shown in their literature to cleave three out of four known mismatches, provided the enzyme level is sufficiently high. One drawback of the Promega kit is that a very large amount of the RNase I enzyme must be used. For detection of single base mismatches, the Promega Technical Manual recommends using approximately 100 times more RNase I than is normally used in the standard RNase detection assay.
For example, in a commercial product marketed by Ambion, Inc. (Austin, Tex.) for mutation detection using the NIRCA.TM. method, two different RNase compositions are provided to be tried and adopted at user discretion. One composition contains a combination of RNase A (approximately 0.5 .mu.g/ml) and RNase I (approximately 175 units/ml), and the other contains RNase A only (approximately 14 .mu.g/ml), where these amounts refer to the recommended final working concentrations.
Despite the favorable characteristics of RNase I, the fact that approximately 100 times as much RNase I as RNase A is needed for an assay procedure has effectively prevented the use of RNase I for detecting base pair mismatches. Of even greater concern is the fact that even when the recommended high levels of RNase I are used, many single-base mismatches are not cleaved using the reaction mixture in the Promega Technical Manual (FIG. 1).
Therefore, a need exists for a sensitive technique that reliably detects point mutations in long target regions of nucleic acids in a safe, easy, cost effective way. Particularly advantageous would be such a method that would lend itself to the use of various RNase enzymes and that would not require radioactively labeled components.